Comparative transcriptomic analysis of circulating endothelial cells in sickle cell stroke.

Ischemic stroke (IS) is one of the most impairing complications of sickle cell anemia (SCA), responsible for 20% of mortality in patients. Rheological alterations, adhesive properties of sickle reticulocytes, leukocyte adhesion, inflammation and endothelial dysfunction are related to the vasculopathy observed prior to ischemic events. The role of the vascular endothelium in this complex cascade of mechanisms is emphasized, as well as in the process of ischemia-induced repair and neovascularization. The aim of the present study was to perform a comparative transcriptomic analysis of endothelial colony-forming cells (ECFCs) from SCA patients with and without IS. Next, to gain further insights of the biological relevance of differentially expressed genes (DEGs), functional enrichment analysis, protein-protein interaction network (PPI) construction and in silico prediction of regulatory factors were performed. Among the 2469 DEGs, genes related to cell proliferation (AKT1, E2F1, CDCA5, EGFL7), migration (AKT1, HRAS), angiogenesis (AKT1, EGFL7) and defense response pathways (HRAS, IRF3, TGFB1), important endothelial cell molecular mechanisms in post ischemia repair were identified. Despite the severity of IS in SCA, widely accepted molecular targets are still lacking, especially related to stroke outcome. The comparative analysis of the gene expression profile of ECFCs from IS patients versus controls seems to indicate that there is a persistent angiogenic process even after a long time this complication has occurred. Thus, this is an original study which may lead to new insights into the molecular basis of SCA stroke and contribute to a better understanding of the role of endothelial cells in stroke recovery.

Role of EGFL7 in human cancers: A review.

EGFL7 is a proangiogenic factor. It has been widely described with having a vital role in tubulogenesis and regulation of angiogenesis, mainly during embryogenesis and organogenesis. It has been mainly associated with NOTCH pathway, but there are reports showing association with MAPK and integrin pathways. Given its association with angiogenesis and these other pathways, there are several studies associating EGFL7 with carcinogenesis. In fact, most of the studies have pointed to EGFL7 as an oncogene, and some of them suggest EGFL7 expression as a possible biomarker of prognosis or use for a patient's follow-up. Here, we review the molecular pathways which EGFL7 is associated and highlight several studies describing the role of EGFL7 in tumorigenesis, separated by tumor type. Besides its role on angiogenesis, EGFL7 may act in other pathways as oncogene, which makes it a possible biomarker and a candidate to targeted therapy.

Gene Expression Profiling between Patient Groups with High and Low Ki67 Levels after Short-term Preoperative Aromatase Inhibitor Treatment for Breast Cancer.

According to a recent report, a low Ki67 level after short-term preoperative hormone therapy (post-Ki67) might suggest a more favorable prognosis compared with a high post-Ki67 level in patients with hormone receptorpositive/human epidermal growth factor 2-negative (HR+/HER2-) breast cancer with high levels of Ki67. This study aimed to evaluate the pre-treatment genetic differences between these two patient groups. Forty-five luminal B-like patients were stratified into two groups, namely, a group with high (H→H) and one with low (H→L) Ki67 levels after short-term preoperative aromatase inhibitor (AI) treatment. We compared pre-treatment gene expression profiles between the two groups. In gene level analysis, there was no significant difference between the two groups by the class comparison test. In pathway analysis, five metabolism-related gene sets were significantly upregulated in the H→L group (p≤0.05). In the search for novel targets, five genes (PARP, BRCA2, FLT4, CDK6, and PDCD1LG2) showed significantly higher expression in the H→H group (p≤0.05). Several metabolism-related pathways were associated with sensitivity to AI. In the future, it will be necessary to seek out new therapeutic strategies for the poor prognostic group with high post-Ki67.

Early-Onset Pulmonary Events with Combined Brigatinib and Afatinib Treatment of L858/cisT790M/cisC797S NSCLC: A Case Report.

BACKGROUND Brigatinib is used for anaplastic lymphoma kinase (ALK)-positive lung cancer treatment, and some research showed it was useful in treating triple-mutant epidermal growth factor receptor lung cancer. Clinical trials have shown some potential pulmonary toxicities of brigatinib. The early-onset pulmonary events (EOPEs) of brigatinib are associated with high dosage and older age. The successful treatment of EOPEs with steroids was reported. We present the case of a patient with epidermal growth factor receptor L858R/cis-T790M/cis-C797S triple mutations who developed EOPEs after using brigatinib together with afatinib, and the patient was successfully treated with high-dose steroids. CASE REPORT A 54-year-old woman with underlying stage IV lung adenocarcinoma, ECOG score of 0, was treated with brigatinib and afatinib due to disease progression secondary to L858R/cis-T790M/cis-C797S triple mutations. After starting brigatinib and afatinib, she developed dyspnea and dry cough within 2 days and was intubated due to hypercapnic respiratory failure. The chest X-ray showed bilateral interstitial infiltrates while chest computed tomography (CT) showed bilateral ground-glass opacities. EOPEs were suspected and methylprednisolone was prescribed. The oxygenation of the patient improved and her chest CT showed complete resolution after 2 weeks of steroid treatment. CONCLUSIONS This is the first reported case in which brigatinib combined with afatinib induced EOPEs in a patient with triple-mutant epidermal growth factor receptors of lung cancer. Use of doubled tyrosine kinase inhibitors may result in increased risk of pulmonary toxicities that require high alertness, and the respiratory symptoms should be monitored closely after prescription. The early treatment of EOPEs with high-dose steroids resulted in remarkable improvement.

Drosophila Homolog of the Human Carpenter Syndrome Linked Gene, MEGF8, Is Required for Synapse Development and Function.

Drosophila multiple epidermal growth factor-like domains 8 (dMegf8) is a homolog of human MEGF8 MEGF8 encodes a multidomain transmembrane protein which is highly conserved across species. In humans, MEGF8 mutations cause a rare genetic disorder called Carpenter syndrome, which is frequently associated with abnormal left-right patterning, cardiac defects, and learning disabilities. MEGF8 is also associated with psychiatric disorders. Despite its clinical relevance, MEGF8 remains poorly characterized; and although it is highly conserved, studies on animal models of Megf8 are also very limited. The presence of intellectual disabilities in Carpenter syndrome patients and association of MEGF8 with psychiatric disorders indicate that mutations in MEGF8 cause underlying defects in synaptic structure and functions. In this study, we investigated the role of Drosophila dMegf8 in glutamatergic synapses of the larval neuromuscular junctions (NMJ) in both males and females. We show that dMegf8 localizes to NMJ synapses and is required for proper synaptic growth. dMegf8 mutant larvae and adults show severe motor coordination deficits. At the NMJ, dMegf8 mutants show altered localization of presynaptic and postsynaptic proteins, defects in synaptic ultrastructure, and neurotransmission. Interestingly, dMegf8 mutants have reduced levels of the Type II BMP receptor Wishful thinking (Wit). dMegf8 displays genetic interactions with neurexin-1 (dnrx) and wit, and in association with Dnrx and Wit plays an essential role in synapse organization. Our studies provide insights into human MEGF8 functions and potentially into mechanisms that may underlie intellectual disabilities observed in Carpenter syndrome as well as MEGF8-related synaptic structural and/or functional deficits in psychiatric disorders.SIGNIFICANCE STATEMENT Carpenter syndrome, known for over a century now, is a genetic disorder linked to mutations in Multiple Epidermal Growth Factor-like Domains 8 (MEGF8) gene and associated with intellectual disabilities among other symptoms. MEGF8 is also associated with psychiatric disorders. Despite the high genetic conservation and clinical relevance, the functions of MEGF8 remain largely uncharacterized. Patients with intellectual disabilities and psychiatric diseases often have an underlying defect in synaptic structure and function. This work defines the role of the fly homolog of human MEGF8, dMegf8, in glutamatergic synapse growth, organization, and function and provide insights into potential functions of MEGF8 in human central synapses and synaptic mechanisms that may underlie psychiatric disorders and intellectual disabilities seen in Carpenter syndrome.

Tyrosine hydroxylase gene promoter activity is upregulated in female catecholaminergic neuroblastoma cells following activation of a Gαq-coupled designer receptor.

Tyrosine hydroxylase is the rate-limiting enzyme of catecholamine biosynthesis that catalyzes the conversion of L-tyrosine to L-3,4-dihydroxyphenylalanine. The tyrosine hydroxylase gene is regulated by extracellular signaling molecules such as epidermal growth factor, nerve growth factor and steroids. Here, we investigated whether the activity of the tyrosine hydroxylase gene promoter is upregulated by activation of G protein-coupled receptors, the largest group of plasma membrane receptors. We used catecholaminergic neuroblastoma cells as a cellular model and chromatin-integrated tyrosine hydroxylase promoter-luciferase reporter genes. The results show that stimulation of Rαq, a Gαq-coupled designer receptor, triggered transcription of a reporter gene driven by the tyrosine hydroxylase promoter. Transcription was attenuated by overexpression of regulator of G-protein signaling-2, which activates the GTPase activity of the G protein α-subunit, and by a truncated, dominant-negative mutant of phospholipase Cß3. Extracellular signal-regulated protein kinase was identified as the signal transducer. At the transcriptional level, tyrosine hydroxylase promoter activity was found to be controlled by the transcription factor CREB. Expression experiments with the adenoviral regulator protein E1A, an inhibitor of CBP/p300 histone acetyltransferases, showed that transcription of the reporter gene controlled by the tyrosine hydroxylase is under epigenetic control. We identified the protein phosphatases MAP kinase phosphatase-1 and calcineurin as part of a shutdown device of the signaling cascade linking Rαq designer receptor activation to tyrosine hydroxylase gene transcription. We conclude that tyrosine hydroxylase promoter activity is controlled by Gαq-coupled receptors.

Effect of NOTCH3 EGFr Group, Sex, and Cardiovascular Risk Factors on CADASIL Clinical and Neuroimaging Outcomes.

BACKGROUND: A retrospective study has shown that EGFr (epidermal growth factor-like repeat) group in the NOTCH3 gene is an important cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) disease modifier of age at first stroke and white matter hyperintensity (WMH) volume. No study has yet assessed the effect of other known CADASIL modifiers, that is, cardiovascular risk factors and sex, in the context of NOTCH3 EGFr group. In this study, we determined the relative disease-modifying effects of NOTCH3 EGFr group, sex and cardiovascular risk factor on disease severity in the first genotype-driven, large prospective CADASIL cohort study, using a comprehensive battery of CADASIL clinical outcomes and neuroimaging markers. METHODS: Patients with CADASIL participated in a single-center, prospective cohort study (DiViNAS [Disease Variability in NOTCH3 Associated Small Vessel Disease]) between 2017 and 2020. The study protocol included a clinical assessment, neuropsychological test battery and brain magnetic resonance imaging on a single research day. Multivariable linear, logistic and Cox regression models were used to cross-sectionally assess the effect of CADASIL modifiers on clinical severity (stroke, disability, processing speed) and neuroimaging markers (WMH volume, peak width of skeletonized mean diffusivity, lacune volume, brain volume, cerebral microbleed count). RESULTS: Two hundred patients with CADASIL participated, of which 103 harbored a NOTCH3 EGFr 1-6 variant and 97 an EGFr 7-34 variant. NOTCH3 EGFr 1-6 group was the most important modifier of age at first stroke (hazard ratio, 2.45 [95% CI, 1.39-4.31]; P=0.002), lacune volume (odds ratio, 4.31 [95% CI, 2.31-8.04]; P=4.0×10-6), WMH volume (B=0.81 [95% CI, 0.60-1.02]; P=1.1×10-12), and peak width of skeletonized mean diffusivity (B=0.65 [95% CI, 0.44-0.87]; P=1.6×10-8). EGFr 1-6 patients had a significantly higher WMH volume in the anterior temporal lobes and superior frontal gyri and a higher burden of enlarged perivascular spaces. After NOTCH3 EGFr group, male sex and hypertension were the next most important modifiers of clinical outcomes and neuroimaging markers. CONCLUSIONS: NOTCH3 EGFr group is the most important CADASIL disease modifier not only for age at first stroke and WMH volume but also strikingly so for a whole battery of clinically relevant disease measures such as lacune volume and peak width of skeletonized mean diffusivity. NOTCH3 EGFr group is followed in importance by sex, hypertension, diabetes, and smoking.

Single-cell RNA sequencing reveals differential expression of EGFL7 and VEGF in giant-cell tumor of bone and osteosarcoma.

Dysregulation of angiogenesis is associated with tumor development and is accompanied by altered expression of pro-angiogenic factors. EGFL7 is a newly identified antigenic factor that plays a role in various cancers such as breast cancer, lung cancer, and acute myeloid leukemia. We have recently found that EGFL7 is expressed in the bone microenvironment, but its role in giant-cell tumor of bone (GCTB) and osteosarcoma (OS) is unknown. The aims of this study are to examine the gene expression profile of EGFL7 in GCTB and OS and compare with that of VEGF-A-D and TNFSF11 using single-cell RNA sequencing data. In-depth differential expression analyses were employed to characterize their expression in the constituent cell types of GCTB and OS. Notably, EGFL7 in GCTB was expressed at highest levels in the endothelial cell (EC) cluster followed by osteoblasts, myeloid cells, and chondrocytes, respectively. In OS, EGFL7 exhibited highest expression in EC cell cluster followed by osteoblastic OS cells, myeloid cells 1, and carcinoma associated fibroblasts (CAFs), respectively. In comparison, VEGF-A is expressed at highest levels in myeloid cells followed by OCs in GCTB, and in myeloid cells, and OCs in OS. VEGF-B is expressed at highest levels in chondrocytes in GCTB and in OCs in OS. VEGF-C is strongly enriched in ECs and VEGF-D is expressed at weak levels in all cell types in both GCTB and OS. TNFSF11 (or RANKL) shows high expression in CAFs and osteoblastic OS cells in OS, and osteoblasts in GCTB. This study investigates pro-angiogenic genes in GCTB and OS and suggests that these genes and their expression patterns are cell-type specific and could provide potential prognostic biomarkers and cell type target treatment for GCTB and OS.

Mice lacking MBNL1 and MBNL2 exhibit sudden cardiac death and molecular signatures recapitulating myotonic dystrophy.

Myotonic dystrophy (DM) is caused by expansions of C(C)TG repeats in the non-coding regions of the DMPK and CNBP genes, and DM patients often suffer from sudden cardiac death due to lethal conduction block or arrhythmia. Specific molecular changes that underlie DM cardiac pathology have been linked to repeat-associated depletion of Muscleblind-like (MBNL) 1 and 2 proteins and upregulation of CUGBP, Elav-like family member 1 (CELF1). Hypothesis solely targeting MBNL1 or CELF1 pathways that could address all the consequences of repeat expansion in heart remained inconclusive, particularly when the direct cause of mortality and results of transcriptome analyses remained undetermined in Mbnl compound knockout (KO) mice with cardiac phenotypes. Here, we develop Myh6-Cre double KO (DKO) (Mbnl1-/-; Mbnl2cond/cond; Myh6-Cre+/-) mice to eliminate Mbnl1/2 in cardiomyocytes and observe spontaneous lethal cardiac events under no anesthesia. RNA sequencing recapitulates DM heart spliceopathy and shows gene expression changes that were previously undescribed in DM heart studies. Notably, immunoblotting reveals a nearly 6-fold increase of Calsequestrin 1 and 50% reduction of epidermal growth factor proteins. Our findings demonstrate that complete ablation of MBNL1/2 in cardiomyocytes is essential for generating sudden death due to lethal cardiac rhythms and reveal potential mechanisms for DM heart pathogenesis.

EGF repeats of epidermal growth factor­like domain 7 promote endothelial cell activation and tumor escape from the immune system.

The tumor blood vessel endothelium forms a barrier that must be crossed by circulating immune cells in order for them to reach and kill cancer cells. Epidermal growth factor­like domain 7 (Egfl7) represses this immune infiltration by lowering the expression levels of leukocyte adhesion receptors on the surface of endothelial cells. However, the protein domains involved in these properties are not completely understood. Egfl7 is structurally composed of the predicted EMI­, EGF­ and C­terminal domains. The present study aimed to investigate the roles of these different domains in tumor development by designing retroviruses coding for deletion mutants and then infecting 4T1 breast cancer cell populations, which consequently overexpressed the variants. By performing in vitro soft­agar assays, it was found that Egfl7 and its deletion variants did not affect cell proliferation or anchorage­independent growth. When 4T1 cells expressing either the wild­type Egfl7 protein or Egfl7 domain variants were implanted in mice, Egfl7 expression markedly promoted tumor development and deletion of the EGF repeats decreased the tumor growth rate. By contrast, deleting any other domain displayed no significant effect on tumor development. The overexpression of Egfl7 also decreased T cell and natural killer cell infiltration in tumors, as determined by immunofluorescence staining of tumor sections, whereas deletion of the EGF repeats inhibited this effect. Reverse transcription­quantitative PCR analysis of the mechanisms involved revealed that deleting the EGF repeats partially restored the expression levels of vascular cell adhesion molecule 1 and E­selectin, which were suppressed by overexpression of Egfl7 in endothelial cells in vitro. This resulted in a higher number of lymphocytes bound to HUVEC expressing Egfl7­ΔEGF compared with HUVEC expressing wild­type Egfl7, as assessed by fluorescent­THP­1 adhesion assays onto endothelial cells. Overall, the present study demonstrated that the EGF repeats may participate in the protumoral and anti­inflammatory effects of Egfl7.

Targeting miR-126 in inv(16) acute myeloid leukemia inhibits leukemia development and leukemia stem cell maintenance.

Acute myeloid leukemia (AML) harboring inv(16)(p13q22) expresses high levels of miR-126. Here we show that the CBFB-MYH11 (CM) fusion gene upregulates miR-126 expression through aberrant miR-126 transcription and perturbed miR-126 biogenesis via the HDAC8/RAN-XPO5-RCC1 axis. Aberrant miR-126 upregulation promotes survival of leukemia-initiating progenitors and is critical for initiating and maintaining CM-driven AML. We show that miR-126 enhances MYC activity through the SPRED1/PLK2-ERK-MYC axis. Notably, genetic deletion of miR-126 significantly reduces AML rate and extends survival in CM knock-in mice. Therapeutic depletion of miR-126 with an anti-miR-126 (miRisten) inhibits AML cell survival, reduces leukemia burden and leukemia stem cell (LSC) activity in inv(16) AML murine and xenograft models. The combination of miRisten with chemotherapy further enhances the anti-leukemia and anti-LSC activity. Overall, this study provides molecular insights for the mechanism and impact of miR-126 dysregulation in leukemogenesis and highlights the potential of miR-126 depletion as a therapeutic approach for inv(16) AML.

Whole-exome sequencing reveals a role of HTRA1 and EGFL8 in brain white matter hyperintensities.

White matter hyperintensities (WMH) are among the most common radiological abnormalities in the ageing population and an established risk factor for stroke and dementia. While common variant association studies have revealed multiple genetic loci with an influence on their volume, the contribution of rare variants to the WMH burden in the general population remains largely unexplored. We conducted a comprehensive analysis of this burden in the UK Biobank using publicly available whole-exome sequencing data (n up to 17 830) and found a splice-site variant in GBE1, encoding 1,4-alpha-glucan branching enzyme 1, to be associated with lower white matter burden on an exome-wide level [c.691+2T>C, ß = -0.74, standard error (SE) = 0.13, P = 9.7 × 10-9]. Applying whole-exome gene-based burden tests, we found damaging missense and loss-of-function variants in HTRA1 (frequency of 1 in 275 in the UK Biobank population) to associate with an increased WMH volume (P = 5.5 × 10-6, false discovery rate = 0.04). HTRA1 encodes a secreted serine protease implicated in familial forms of small vessel disease. Domain-specific burden tests revealed that the association with WMH volume was restricted to rare variants in the protease domain (amino acids 204-364; ß = 0.79, SE = 0.14, P = 9.4 × 10-8). The frequency of such variants in the UK Biobank population was 1 in 450. The WMH volume was brought forward by ∼11 years in carriers of a rare protease domain variant. A comparison with the effect size of established risk factors for WMH burden revealed that the presence of a rare variant in the HTRA1 protease domain corresponded to a larger effect than meeting the criteria for hypertension (ß = 0.26, SE = 0.02, P = 2.9 × 10-59) or being in the upper 99.8% percentile of the distribution of a polygenic risk score based on common genetic variants (ß = 0.44, SE = 0.14, P = 0.002). In biochemical experiments, most (6/9) of the identified protease domain variants resulted in markedly reduced protease activity. We further found EGFL8, which showed suggestive evidence for association with WMH volume (P = 1.5 × 10-4, false discovery rate = 0.22) in gene burden tests, to be a direct substrate of HTRA1 and to be preferentially expressed in cerebral arterioles and arteries. In a phenome-wide association study mapping ICD-10 diagnoses to 741 standardized Phecodes, rare variants in the HTRA1 protease domain were associated with multiple neurological and non-neurological conditions including migraine with aura (odds ratio = 12.24, 95%CI: 2.54-35.25; P = 8.3 × 10-5]. Collectively, these findings highlight an important role of rare genetic variation and the HTRA1 protease in determining WMH burden in the general population.

Epigenetic down-regulation of microRNA-126 in scleroderma endothelial cells is associated with impaired responses to VEGF and defective angiogenesis.

Impaired angiogenesis in scleroderma (SSc) is a critical component of SSc pathology. MicroRNA-126 (miR-126) is expressed in endothelial cells (MVECs) where it regulates VEGF responses by repressing the negative regulators of VEGF, including the sprouty-related protein-1 (SPRED1), and phosphoinositide-3 kinase regulatory subunit 2 (PIK3R2). MVECs were isolated from SSc skin and matched subjects (n = 6). MiR-126 expression was measured by qPCR and in situ hybridization. Matrigel-based tube assembly was used to test angiogenesis. MiR-126 expression was inhibited by hsa-miR-126 inhibitor and enhanced by hsa-miR-126 Mimic. Epigenetic regulation of miR-126 expression was examined by the addition of epigenetic inhibitors (Aza and TSA) to MVECs and by bisulphite genomic sequencing of DNA methylation of the miR-126 promoter region. MiR-126 expression, as well as EGFL7 (miR-126 host gene), in SSc-MVECs and skin, was significantly down-regulated in association with increased expression of SPRED1 and PIK3R2 and diminished response to VEGF. Inhibition of miR-126 in NL-MVECs resulted in reduced angiogenic capacity, whereas overexpression of miR-126 in SSc-MVECs resulted in enhanced tube assembly. Addition of Aza and TSA normalized miR-126 and EGFL7 expression levels in SSc-MVECs. Heavy methylation in miR-126/EGFL7 gene was noted. In conclusion, these results demonstrate that the down-regulation of miR-126 results in impaired VEGF responses.

An increase in elastogenic components in the placental villi of women with chronic venous disease during pregnancy is associated with decreased EGFL7 expression level.

Chronic venous disease (CVD) is the response to a series of hemodynamic changes in the venous system and the onset of this disease is often triggered by pregnancy. Placental tissue is particularly sensitive to the characteristic changes which occurs in venous hypertension. In this regard, changes in the extracellular matrix (ECM), that occur to adapt to this situation, are fundamental to controlling elastogenesis. Therefore, the aim of the present study was to analyze the changes that occur in the mRNA and protein expression level of proteins related to elastogenesis in the placental villi of women diagnosed with CVD, in the third trimester of pregnancy. An observational, analytical and prospective cohort study was conducted, in which the placenta from 62 women with CVD were compared with that in placenta from 52 women without a diagnosis of CVD. Gene and protein expression levels were analyzed using reverse transcription­quantitative PCR and immunohistochemistry, respectively. The results showed a significant decrease in the gene and protein expression level of EGFL7 in the placental villi of women with CVD. By contrast, significant increases in the gene and protein expression level of ECM­related proteins, such as tropoelastin, fibulin 4, fibrillin 1 and members of the lysyl oxidase family (LOX and LOXL­1) were also found in the placental villi of women with CVD. To the best of our knowledge, the results from the present study showed for the first time that CVD during pregnancy was associated with changes in the mRNA and protein expression level in essential components of the EGFL7­modulated elastogenesis process in placental villi.

Self-renewal of double-negative 3 early thymocytes enables thymus autonomy but compromises the ß-selection checkpoint.

T lymphocyte differentiation in the steady state is characterized by high cellular turnover whereby thymocytes do not self-renew. However, if deprived of competent progenitors, the thymus can temporarily maintain thymopoiesis autonomously. This bears a heavy cost, because prolongation of thymus autonomy causes leukemia. Here, we show that, at an early stage, thymus autonomy relies on double-negative 3 early (DN3e) thymocytes that acquire stem-cell-like properties. Following competent progenitor deprivation, DN3e thymocytes become long lived, are required for thymus autonomy, differentiate in vivo, and include DNA-label-retaining cells. At the single-cell level, the transcriptional programs of thymopoiesis in autonomy and the steady state are similar. However, a new cell population emerges in autonomy that expresses an aberrant Notch target gene signature and bypasses the ß-selection checkpoint. In summary, DN3e thymocytes have the potential to self-renew and differentiate in vivo if cell competition is impaired, but this generates atypical cells, probably the precursors of leukemia.

Schwann cell plasticity regulates neuroblastic tumor cell differentiation via epidermal growth factor-like protein 8.

Adult Schwann cells (SCs) possess an inherent plastic potential. This plasticity allows SCs to acquire repair-specific functions essential for peripheral nerve regeneration. Here, we investigate whether stromal SCs in benign-behaving peripheral neuroblastic tumors adopt a similar cellular state. We profile ganglioneuromas and neuroblastomas, rich and poor in SC stroma, respectively, and peripheral nerves after injury, rich in repair SCs. Indeed, stromal SCs in ganglioneuromas and repair SCs share the expression of nerve repair-associated genes. Neuroblastoma cells, derived from aggressive tumors, respond to primary repair-related SCs and their secretome with increased neuronal differentiation and reduced proliferation. Within the pool of secreted stromal and repair SC factors, we identify EGFL8, a matricellular protein with so far undescribed function, to act as neuritogen and to rewire cellular signaling by activating kinases involved in neurogenesis. In summary, we report that human SCs undergo a similar adaptive response in two patho-physiologically distinct situations, peripheral nerve injury and tumor development.

Descriptive and functional characterization of epidermal growth factor­like domain 8 in mouse cortical thymic epithelial cells by integrated analysis of gene expression signatures and networks.

Epidermal growth factor­like domain 8 (EGFL8), a newly identified member of the EGFL family, and plays negative regulatory roles in mouse thymic epithelial cells (TECs) and thymocytes. However, the role of EGFL8 in these cells remains poorly understood. In the present study, in order to characterize the function of EGFL8, genome­wide expression profiles in EGFL8­overexpressing or ­silenced mouse cortical TECs (cTECs) were analyzed. Microarray analysis revealed that 458 genes exhibited a >2­fold change in expression levels in the EGFL8­overexpressing vs. the EGFL8­silenced cTECs. Several genes involved in a number of cellular processes, such as the cell cycle, proliferation, growth, migration and differentiation, as well as in apoptosis, reactive oxygen species generation, chemotaxis and immune responses, were differentially expressed in the EGFL8­overexpressing or ­silenced cTECs. WST­1 analysis revealed that that the overexpression of EGFL8 inhibited cTEC proliferation. To investigate the underlying mechanisms of EGFL8 in the regulation of cTEC function, genes related to essential cellular functions were selected. Reverse transcription­polymerase chain reaction analysis revealed that EGFL8 knockdown upregulated the expression of cluster differentiation 74 (CD74), Fas ligand (FasL), C­X­C motif chemokine ligand 5 (CXCL5), CXCL10, CXCL16, C­C motif chemokine ligand 20 (CCL20), vascular endothelial growth factor­A (VEGF­A), interferon regulatory factor 7 (Irf7), insulin­like growth factor binding protein­4 (IGFBP­4), thrombospondin 1 (Thbs1) and nuclear factor κB subunit 2 (NF­κB2) genes, and downregulated the expression of angiopoietin­like 1 (Angptl1), and neuropilin­1 (Nrp1) genes. Additionally, EGFL8 silencing enhanced the expression of anti­apoptotic molecules, such as B­cell lymphoma­2 (Bcl­2) and Bcl­extra large (Bcl­xL), and that of cell cycle­regulating molecules, such as cyclin­dependent kinase 1 (CDK1), CDK4, CDK6 and cyclin D1. Moreover, gene network analysis revealed that EGFL8 exerted negative effects on VEGF­A gene expression. Hence, the altered expression of several genes associated with EGFL8 expression in cTECs highlights the important physiological processes in which EGFL8 is involved, and provides insight into its biological functions.

Suppression of lncRNA HOTAIR alleviates RCC angiogenesis through regulating miR-126/EGFL7 axis.

Renal cell carcinoma (RCC) has the highest mortality rate among urological cancers and tumor angiogenesis that plays a critical role in RCC progress. Epidermal growth factor-like domain multiple 7 (EGFL7) has been recently identified as a regulator in RCC tumor angiogenesis and progression. Long noncoding RNA (LncRNA) HOTAIR has been considered as a pro-oncogene in multiple cancers, but its precise mechanism of tumor angiogenesis has rarely been reported. MicroRNA-126 (miR-126) functions as a tumor suppressor in RCC. However, the underlying tumor angiogenesis mechanism of HOTAIR/miR-126 axis in RCC has not been studied. The proliferation, migration, angiogenesis, and expression of EGFL7 and related proteins in extracellular signal-regulated kinase (ERK)/activators of transcription 3 (STAT3) signal pathway were determined to examine the effect and mechanism of HOTAIR and miR-126 on RCC progress. The regulatory relationship of HOTAIR and miR-126, as well as miR-126 and EGFL7 were tested using dual-luciferase reporter assay. Aenograft RCC mice model was used to examine the effect of HOTAIR on RCC tumor growth and metastasis in vivo. HOTAIR knockdown and miR-126 overexpression suppressed the proliferation, migration, and angiogenesis of RCC cells. HOTAIR regulated EGFL7 expression by competitively binding to miR-126. Knockdown of HOTAIR significantly suppressed the RCC tumor progression and lung metastasis in vivo. These findings suggest that lncRNA HOTAIR regulate RCC angiogenesis through miR-126/EGFL7 axis and provide a new perspective on the molecular pathways of angiogenesis in RCC development, which might be potential therapeutic targets for RCC treatment.

Prevention of age-associated neuronal hyperexcitability with improved learning and attention upon knockout or antagonism of LPAR2.

Recent studies suggest that synaptic lysophosphatidic acids (LPAs) augment glutamate-dependent cortical excitability and sensory information processing in mice and humans via presynaptic LPAR2 activation. Here, we studied the consequences of LPAR2 deletion or antagonism on various aspects of cognition using a set of behavioral and electrophysiological analyses. Hippocampal neuronal network activity was decreased in middle-aged LPAR2-/- mice, whereas hippocampal long-term potentiation (LTP) was increased suggesting cognitive advantages of LPAR2-/- mice. In line with the lower excitability, RNAseq studies revealed reduced transcription of neuronal activity markers in the dentate gyrus of the hippocampus in naïve LPAR2-/- mice, including ARC, FOS, FOSB, NR4A, NPAS4 and EGR2. LPAR2-/- mice behaved similarly to wild-type controls in maze tests of spatial or social learning and memory but showed faster and accurate responses in a 5-choice serial reaction touchscreen task requiring high attention and fast spatial discrimination. In IntelliCage learning experiments, LPAR2-/- were less active during daytime but normally active at night, and showed higher accuracy and attention to LED cues during active times. Overall, they maintained equal or superior licking success with fewer trials. Pharmacological block of the LPAR2 receptor recapitulated the LPAR2-/- phenotype, which was characterized by economic corner usage, stronger daytime resting behavior and higher proportions of correct trials. We conclude that LPAR2 stabilizes neuronal network excitability upon aging and allows for more efficient use of resting periods, better memory consolidation and better  performance in tasks requiring high selective attention. Therapeutic LPAR2 antagonism may alleviate aging-associated cognitive dysfunctions.